The recognition of antigen by T cells is a seminal event in the induction of a specific immune response. The TCR recognizes a peptide bound to an MHC molecule (p/MHC) on the surface of an antigen presenting cell. The TCR is a highly intricate receptor, but has a low affinity for the p/MHC ligand. The basis for the exquisite TCR specificity, despite the low affinity, is still not known. A major advance in our understanding of how T cells recognize antigen has been the direct demonstration of the formation of an immunological synapse. The immunological synapse is a supramolecular structure at the interface between the T cell and a single APC that is stable for many hours. The immunological synapse provides the molecular machinery for integration of p/MHC quality and quantity. These studies have provided a model explaining how a TCR can distinguish between closely related ligands, but many questions remain. In this proposal, our overall goal is to understand how mature T cells and thymocytes can respond to small kinetic and structural changes in the p/MHC ligands. Our laboratory has developed and utilized the Hb/I-Ek antigen system. We have generated and characterized a wide range of T cells, ligands, and transgenic mice for two T cells, 3.L2 and 2.102. Through an integrated investigation of the 3.L2 and 2.102 T cells and ligands we propose to explore several key issues including: 1) how do the TCR binding properties to a continuum of different strength ligands relate to their structure and biological effects, 2) how does the recognition by a single TCR of syngeneic and allogeneic ligands compare, 3) how do immunological synapses in thymocytes compare to those of mature T cells, and 4) what is the role of CD4 in immunological synapse formation and TCR:p/MHC interactions? These proposed studies will provide key molecular details of the interaction between a TCR and the p/MHC ligand. These studies could lead to the development of novel pharmaceuticals which could control unwanted immune responses, such as an autoimmune response.